The long term objective of the proposed research is to understand the molecular mechanisms of genomic instability associated with aging and aging-related diseases. Accelerated telomeric DNA loss occurs in aging-related diseases and after exposure to some environmental DNA damaging agents. Lack of the DNA repair protein WRN accelerates telomere loss and causes the human progeroid Werner syndrome (WS) in which patients prematurely develop multiple aging-related pathologies. The specific goals of this proposal are 1) to determine the molecular mechanisms of telomeric DNA loss associated with DNA damaging agents and WS, and 2) to define the roles for the repair protein WRN in telomere preservation. The hypothesis to be tested is that damage directly to telomeric DNA contributes to telomere attrition, and that WRN protein preserves telomeres by preventing and/or repairing breaks in telomeric DNA. A shuttle vector mutagenesis assay that measures mutations in a defined target will be used to examine the role of WRN protein in preventing replication-induced telomeric DNA deletions that may occur either spontaneously or after exposure to the environmental mutagen chromium (VI). Exposure to Cr(VI) is associated with respiratory cancers, and induces replication-blocking adducts and breaks in DNA sequences that are prevalent in telomeres. Thus, Cr(VI) is an excellent model for investigating the consequences of environmental DNA damage on telomeric DNA replication. The shuttle vector approach allows for analysis of independent and rare mutation events and eliminates selective pressure against the loss of telomeric repeats. To investigate a role for WRN in repairing breaks at telomeres directly, confocal microscopy will be used to induce DNA double strand breaks selectively at telomeres with a UVA laser in live cells. The broad goals of this proposal are to determine the impact of DNA damage and environmental mutagens on the integrity of telomeric DNA in chromosome ends, and to examine cellular pathways for repairing telomeres. Given the critical role for telomeres in aging and cancer, a mechanistic understanding of the genetic and environmental factors that accelerate telomeric DNA loss should aid in the identification of risk factors for premature aging. Identifying mechanisms of telomere loss and cellular processes that preserve telomeric DNA is crucial for the design of intervention therapies that prevent or delay the onset of diseases associated with aging and cancer.